禽呼肠孤病毒σA和σNS蛋白激活PI3K/Akt信号通路的研究

The Study of the Activation of PI3K/Akt Pathway by the σA and σNS Protein of Avian Reovirus

旨在探讨禽呼肠孤病毒(ARV)的σA和σNS蛋白是否是通过PXXP或YXXXM基序来激活PI3K/Akt信号通路的。作者采用重叠延伸PCR的方法,将σA和σNS基因的PXXP或YXXXM基序进行突变后构建了重组质粒,并在Vero细胞中进行了表达。通过流式细胞术和Western blot,检测转染后Vero细胞磷酸化Akt(P-Akt)的表达量,并与野生型σA和σNS蛋白激活的P-Akt表达量进行比较。结果显示,σA和σNS基因均得到了表达,σA基因的110—114和114—117位的PXXP基序突变后,Vero细胞P-Akt的表达量明显下降。可见,ARV的σA蛋白,是通过PXXP基序来激活PI3K/Akt信号通路的。本研究从细胞信号转导角度揭示了ARV与宿主相互作用的机制,也为寻找抗ARV药物作用靶标提供了新的思路。

The aim of the present study was to find out whether ARV activates the phosphatidylinositol 3-Kinase-dependent Akt（PI3K/Akt）pathway according to the PXXP or YXXXM motif ofσA andσNS protein.Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motif ofσAandσNSgene.Recombined plasmids that contain mutantσAandσNSgenes were generated.The Akt phosphorylation profile of transfected cells were examined by flow cytometry and Western blot.The results showed thatσAandσNSgenes were expressed in the Vero cells,and the expression of P-Akt of theσA mutant groups（Amino acid 110-114 and 114-117）decreased markedly.The results indicated that theσA protein of ARV activate the PI3K/Akt pathway by the PXXP motif.The results of this study reveal the mechanisms by which ARV manipulate the cellular signal transduction pathways,which may provide new ideas for novel drug targets.